Assays for use in a physician's office or at home by unskilled personnel are needed. Assays suitable for such use are desirably (1) rapid, (2) easy to perform and tolerant of the deviations from precise procedures, and (3) readable without expensive instrumentation. Many companies have introduced test kits to screen for Group A Streptococcus in the physician's office. Other companies have introduced kits for home use to detect pregnancy and kits to detect ovulation.
Some of these kits are enzyme immunoassays where the formation of a colored species indicates the presence (or absence) of the analyte. In the hands of skilled laboratory technicians these tests work well. However, physician's office personnel (who may or may not be trained and who are frequently interrupted in their work) and untrained home users have experienced difficulty using these kits. The enzyme immunoassays require multiple incubations for precisely timed periods and precision in pipetting very small amounts of reagents. Frequently these requirements cannot be met.
Other kits presently available are agglutination immunoassays, typically using latex or erythrocytes as supports. Here again, while the assays may work adequately when used by skilled laboratory technicians, physician's office personnel and home users experience difficulty. Agglutination assays are difficult to read at low analyte concentrations and they suffer sometimes from non-specific agglutination.
Efforts have been made to develop assays which require fewer manipulations and which can be read without expensive instrumentation. South African Pat. No. 84/9397 to Campbell et al. (issued July 31, 1985 and corresponding to allowed U.S. Ser. No. 579,667) describes a solid phase assay with a visible readout. The word "visible" as used in that patent and herein means the label can be detected visually without the use of expensive instrumentation. The system described in Campbell et al. includes a solid support having a binder supported on a test area. It also has a tracer that has a visible label. The binder is present on the support in a concentration whereby the tracer when bound to the support under assay conditions (through the binder or through the analyte which is bound to the binder) is visible. The preferred material for the solid support is nitrocellulose. The patent particularly recommends selecting a material having a pore size such that the tracer when bound remains on the surface of the support. It states that good results have been obtained with a nitrocellulose support having a pore size of from 0.2 to 0.45 microns. The patent goes on to recommend using supports having a large surface area so that the concentration of the binder on the support can be increased. The preferred tracer is a ligand bound to a particulate label. The preferred particulate label is a liposome sac having a dye or other colored substance as marker. The examples describe several assays. One is a pregnancy test having two incubations of one hour each and multiple pipetting and rinsing steps. Another is a tetanus toxoid test with a single one hour incubation. A third is a digoxin assay having two incubations, one for ten minutes and the second for fifteen minutes. The digoxin assay has multiple pipetting and rinsing steps.
Another effort to provide an assay with simplified processing and a visible readout is described in U.S. Pat. No. 4,446,232 to Liotta. That patent describes a device for determining antigens by application of a fluid sample. The device has three layers which comprise two zones. The first zone has a layer containing immobilized antigen and a layer containing absorbed enzyme linked antibodies. The second zone contains materials necessary to react with the enzyme to produce a color. The three layers are assembled with the immobilized antigen layer sandwiched between the enzyme linked antibody layer and the color forming reagent layer. Upon application of a liquid sample containing antigen to the enzyme linked antibody layer, any antigen present reacts with the antibody. The liquid also washes the enzyme linked antibody into the immobilized antigen layer. Any antibody that is saturated with antigen from the sample cannot react with the immobilized antigen and is therefore free to flow through to the second zone where the enzyme reacts with materials in the second zone to produce a color. The device proposed by Liotta reduces the number of technician manipulations but is nonetheless sensitive to variations in technique. Sufficient time must elapse for antigen from the sample to saturate the antibody binding sites and any unsaturated antibody must remain in the immobilized antigen layer long enough to react with the immobilized antigen. Variation in sample amount can cause variations in the rate at which the enzyme linked antibody is washed into the second zone. Additionally, variation in the amount of time between adding the test sample and a stop solution can cause variations in the intensity of color developed thereby making any quantitative comparisons to reference solutions of questionable value.
Another flow through device is shown in U.S. Pat. No. 4,632,901 to Valkirs. In that device antigen is immobilized on a first porous member or membrane. Liquid reagents are applied to one surface of the first membrane. The device also includes a second absorbent member in capillary communication with the surface of the first member opposite the surface where reagents are applied. The second absorbent member has capillary pathways aligned generally transverse to its upper and lower surfaces. The capillary pore size is selected to induce flow of liquid through the first member. The patent indicates that choice of material for the absorbent second member is not critical. Use of cellulose acetate fibers arranged as in a cigarette filter is mentioned. The patent also describes a porous third member which does not bind antibody non-specifically and is interposed between the first and second members.
Yet another patent describing flow through devices is U.S. Pat. No. 4,366,241 to Tom et al. The devices described in that patent have an immunosorbing zone and a liquid absorbing zone in liquid receiving relationship with the immunosorbing zone. The immunosorbing zone has one member of an immunological pair non-diffusively bound to it. At col. 15, lines 5-26, the patent describes materials suitable for use as the immunosorbing zone: "Included among materials which may find use are polysaccharides, e.g. cellulose materials, such as paper and cellulose acetate; silica, inorganic materials such as deactivated alumina, diatomaceous earth, MgSO.sub.4 or other inorganic finely divided material conveniently and substantially uniformly dispersed in a porous polymer matrix, with polymers such as vinyl chloride, vinyl chloride-propylene copolymer and vinyl chloride-vinyl acetate copolymer; cloth, both naturally occurring, e.g., cotton and synthetic, e.g., nylon cloth; porous gels, e.g., silica gel, agarose, dextran, and gelatin; polymeric films, e.g., polyacrylamide; or the like. The important features of the assay device material are that they are able to absorb liquid, particularly aqueous solutions, without substantially impeding the movement of the solutes employed in the assay. In effect, the materials are bibulous; they are porous and allow the flow of the solution; the materials for the immunosorbent layers are preferably non chromatographic; they have reasonable strength or strength can be imparted by means of a support, and they do not interfere with production of the signal by the signal generator."
The Tom et al. patent describes many different assay formats and protocols and different constructions. At col. 16, lines 11-16, it indicates that "one or more additional layers may be involved, particularly between the mip supported conjugate layer [the patent defines a "mip" as a member of an immunological pair] and the lower layer. These layers may serve as barriers to inhibit migration of components of the signal producing system from the lower to the upper layer; as fillers; for flow control; or the like." A three layer immunoabsorbent zone construction is described at col. 16, lines 30-38. In that construction a mip containing layer has relatively low resistance to liquid flow. It is backed by a second layer which is substantially more resistive to liquid flow. The second layer is followed by a porous layer which does not resist flow.
The Tom et al. patent also describes a variety of signal producing systems including chromogens (compounds that absorb light in a distinctive range and compounds that when irradiated emit light of a particular wave length). Where absorptive dyes are used, the patent recommends binding multiple functionalities to the mip, using an absorptive dye with an intense color, and using a substantially transparent immunoabsorbing zone having a sufficient thickness to achieve a reasonable depth of immuno binding signal. It recognizes however that for the most part, the use of dyes, which are measured by light absorption, as the signal label will not provide for the desired sensitivity.
While the devices described above have made progress in the task of developing a device suitable for use by untrained personnel, they all suffer from limitations. Some require procedures too long for screening samples while the patient waits in the doctor's office. Others are not readable at low analyte concentration without the aid of instrumentation. And others require assay procedures too complex for unskilled users.